Effects of Photobiomodulation Therapy on the Expression of Hypoxic Inducible Factor, Vascular Endothelial Growth Factor, and Its Specific Receptor: A Randomized Control Trial in Patients with Diabetic Foot Ulcer.

Background: Impaired angiogenesis is a significant factor contributing to delayed healing in diabetic foot ulcers (DFUs) due to inadequate oxygenation. Objective: This study aimed to investigate the impact of photobiomodulation (PBM) using a Ga-As laser on the release of serum hypoxia-inducible factor 1-α (HIF-1α), vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor-2, and nitric oxide (NO) in diabetic patients with DFUs. Materials and methods: In this double-blind RCT, a total of 30 patients with grade II DFUs were enrolled. The patients were randomly divided into two groups: the PBM (n = 15) and the placebo (n = 15). In the PBM group, a Ga-As laser (904 nm, 2 J/cm2, 90 W) was given for 3 days/week for 4 weeks (11 sessions). In the placebo group, the power was turned off. Both groups received similar standard wound care. Before and after interventions, the levels of serum HIF-1α, VEGF, NO, and sVEGFR-2 were measured. In addition, the percentage decrease in the wound surface area (%DWSA) was measured. Results: Following the intervention, the results revealed that the PBM group had significantly lower levels of VEGF than the placebo group (p = 0.005). The %DWSA was significantly higher in the PBM group compared to the placebo group (p = 0.003). Moreover, VEGF showed a significant negative correlation with %DWSA (p < 0.001). Conclusions: The observed decrease in serum levels of VEGF and an increase in %DWSA, compared to the placebo group, suggests that PBM effectively improves angiogenesis. Furthermore, the significant correlation found between VEGF levels and %DWSA emphasizes the importance of evaluating wound surface in patients as a dependable indicator of enhanced wound angiogenesis. Clinical Trial Registration: NCT02452086.

Underlying Mechanism of Traditional Herbal Formula Chuang-Ling-Ye in the Treatment of Diabetic Foot Ulcer through Network Pharmacology and Molecular Docking.

BACKGROUND: Chuang-Ling-Ye (CLY) has been clinically proven to be an effective Chinese medicine for the treatment of diabetic foot ulcers (DFU). OBJECTIVES: This study aimed to investigate the possible mechanism of CLY in relation to DFU using network pharmacology and molecular docking. MATERIALS AND METHODS: Firstly, relevant targets of CLY against DFU were obtained from TCMSP, Swiss Target Prediction database and GEO database. Then, topological analysis was employed by Cytoscape to screen the top 6 core active ingredients and the top 8 hub targets. Furthermore, the OmicShare Tools were applied for gene ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment analysis. Finally, the results of network pharmacology were verified by molecular docking method. RESULTS: CLY has 61 active compounds and 361 targets after de-duplication, and the top 8 hub targets were EGFR, TP53, CCND1, IL-1B, CREBBP, AR, PTGS2 and PGR. GO enrichment analysis is mainly related to signal transducer activity, receptor activity, and molecular transducer activity. KEGG pathway analysis indicated that these shared targets were primarily focused on AGE-RAGE signaling pathway in diabetic complications, HIF-1 signaling pathway, IL-17 signaling pathway, and JAK-STAT signaling pathway. Molecular docking results showed that physciondiglucoside, 2-cinnamoyl-glucose and kinobeon A were well bound with EGFR, IL-1B, AR and PTGS2. CONCLUSION: This study demonstrated that CLY has anti-oxidative stress and anti-inflammatory effects in the treatment of DFU through various constituents, multiple targets, and multiple pathways, which provides a valuable point of reference for future investigations on CLY.

Integrated analysis of tRNA-derived small RNAs reveals new therapeutic genes of hyperbaric oxygen in diabetic foot ulcers.

Background: To reveal the alterations of tRNA-derived small RNA (tsRNA) expression profiles induced by hyperbaric oxygen (HBO) treatment in diabetic foot ulcers (DFUs) and investigate new therapeutic targets. Materials & methods: tsRNA sequencing was employed in normal skin tissue, in DFUs, and after HBO treatment groups. A quantitative real-time PCR was used to validate tsRNA sequencing results and their targets levels. Bioinformatics analysis was performed to reveal their therapeutic functions in DFUs. Results: A total of 22 tsRNAs were differentially expressed in the three groups. Three selected tsRNAs were validated by quantitative real-time PCR for further analysis, which were all significantly overexpressed in DFU while being normally expressed after HBO treatment. Bioinformatics analysis disclosed that these tsRNAs may play therapeutic roles through the regulation of the Wnt signaling pathway. Conclusion: tsRNAs may be novel useful targets for HBO to treat DFUs.

Hyperbaric oxygen enhances collagen III formation in wound of ZDF rat.

Diabetic foot ulcer (DFU) is a serious complication of diabetes and hyperbaric oxygen therapy (HBOT) is also considered in comprehensive treatment. The evidence supporting the use of HBOT in DFU treatment is controversial. The aim of this work was to introduce a DFU model in ZDF rat by creating a wound on the back of an animal and to investigate the effect of HBOT on the defect by macroscopic evaluation, quantitative histological evaluation of collagen (types I and III), evaluation of angiogenesis and determination of interleukin 6 (IL6) levels in the plasma. The study included 10 rats in the control group (CONT) and 10 in the HBOT group, who underwent HBOT in standard clinical regimen. Histological evaluation was performed on the 18th day after induction of defect. The results show that HBOT did not affect the macroscopic size of the defect nor IL6 plasma levels. A volume fraction of type I collagen was slightly increased by HBOT without reaching statistical significance (1.35+/-0.49 and 1.94+/-0.67 %, CONT and HBOT, respectively). In contrast, the collagen type III volume fraction was ~120 % higher in HBOT wounds (1.41+/-0.81 %) than in CONT ones (0.63+/-0.37 %; p=0.046). In addition, the ratio of the volume fraction of both collagens in the wound ((I+III)w) to the volume fraction of both collagens in the adjacent healthy skin ((I+III)h) was ~65 % higher in rats subjected to HBOT (8.9+/-3.07 vs. 5.38+/-1.86 %, HBOT and CONT, respectively; p=0.028). Vessels density (number per 1 mm2) was found to be higher in CONT vs. HBOT (206.5+/-41.8 and 124+/-28.2, respectively, p<0.001). Our study suggests that HBOT promotes collagen III formation and decreases the number of newly formed vessels at the early phases of healing.

Molecular Biomarkers of Oxygen Therapy in Patients with Diabetic Foot Ulcers.

Hyperbaric oxygen therapy (HBOT) and topical oxygen therapy (TOT) including continuous diffuse oxygen therapy (CDOT) are often utilized to enhance wound healing in patients with diabetic foot ulcerations. High pressure pure oxygen assists in the oxygenation of hypoxic wounds to increase perfusion. Although oxygen therapy provides wound healing benefits to some patients with diabetic foot ulcers, it is currently performed from clinical examination and imaging. Data suggest that oxygen therapy promotes wound healing via angiogenesis, the creation of new blood vessels. Molecular biomarkers relating to tissue inflammation, repair, and healing have been identified. Predictive biomarkers can be used to identify patients who will most likely benefit from this specialized treatment. In diabetic foot ulcerations, specifically, certain biomarkers have been linked to factors involving angiogenesis and inflammation, two crucial aspects of wound healing. In this review, the mechanism of how oxygen works in wound healing on a physiological basis, such as cell metabolism and growth factor signaling transduction is detailed. Additionally, observable clinical outcomes such as collagen formation, angiogenesis, respiratory burst and cell proliferation are described. The scientific evidence for the impact of oxygen on biomolecular pathways and its relationship to the outcomes in clinical research is discussed in this narrative review.

Hyperbaric oxygen potentiates diabetic wound healing by promoting fibroblast cell proliferation and endothelial cell angiogenesis.

BACKGROUND: Diabetic foot ulcer (DFU), one of the diabetic complications, brings high burden to diabetic patients. Hyperbaric oxygen therapy (HBOT) has been proven to be an effective clinical method for the treatment of DFU. However, the mechanisms still to be elucidated. METHODS: Diabetic foot mice model was established, and treated with hyperbaric oxygen. Haematoxylin & eosin (H&E) staining and Masson's trichrome staining were used for the analysis of wound healing. Human skin fibroblast (HSF) and human umbilical vein endothelial cell (HUVECS) were exposed to high glucose and hyperbaric oxygen for studying the mechanism of hyperbaric oxygen promoted wound healing in vitro. Wound healing assay, reactive oxygen species (ROS) assay, cell proliferation assay and tube formation assay were used for the analysis of wound healing. Quantitative-polymerase chain reaction (Q-PCR), Western blotting and enzyme-linked immunosorbent assay (ELISA) were used for the analysis of gene expression. RESULTS: HBOT facilitated wound healing in DFU mice model, and promoted the expression of HIF-1α, NF-κB, VEGFA, SDF-1, VEGFR2 and CXCR4. Hyperbaric oxygen promoted the proliferation, migration and ROS production, as well as the expression of SDF-1 and VEGFA in HSF. HBOT stimulated the proliferation, migration and tube formation, as well as the expression of CXCR4 and VEGFR2 in HUVECS. CONCLUSION: Hyperbaric oxygen potentiates angiogenesis and diabetic wound healing by activating HIF-1α signaling, so as to promote the expression of VEGF/SDF-1 in HSF and the expression of VEGFR/CXCR4 in HUVECS, ultimately to promote the proliferation of HSF and the angiogenesis of HUVECS.

An Overview of Ozone Therapy for Treating Foot Ulcers in Patients With Diabetes.

Diabetic foot ulcer (DFU) is one of the most common and severe complications of diabetes mellitus, which is becoming increasingly prevalent throughout the world, with high mortality and morbidity. Because of the complex pathophysiological processes involved, DFU is difficult to treat effectively with traditional therapies. Ozone therapy, an emerging method, has been reported as potentially beneficial for closure of DFUs and may gradually move to the forefront of clinical practice. Possible mechanisms of action include antioxidant capacity, pathogen inactivation, vascular and endogenous growth factor modulation, and immune system activation. However, some researchers are skeptical about its safety, and clinical trials are lacking. This article reviews the current research and application of ozone therapy for DFUs.

Hyperbaric oxygen therapy accelerates wound healing in diabetic mice by decreasing active matrix metalloproteinase-9.

Diabetic foot ulcers are characterized by hypoxia. For many patients, hyperbaric oxygen (HBO) therapy is the last recourse for saving the limb from amputation, for which the molecular basis is not understood. We previously identified the active form of matrix metalloproteinase-9 (MMP-9) as responsible for diabetic foot ulcer's recalcitrance to healing. Transcription of mmp-9 to the inactive zymogen is upregulated during hypoxia. Activation of the zymogen is promoted by proteases and reactive oxygen species (ROS). We hypothesized that the dynamics of these two events might lead to a lowering of active MMP-9 levels in the wounded tissue. We employed the full-thickness excisional db/db mouse model to study wound healing, and treated the mice to 3.0 atm of molecular oxygen for 90 minutes, 5 days per week for 10 days in an HBO research chamber. Treatment with HBO accelerated diabetic wound healing compared to untreated mice, with more completed and extended reepithelialization. We imaged the wounds for ROS in vivo with a luminol-based probe and found that HBO treatment actually decreases ROS levels. The levels of superoxide dismutase, catalase, and glutathione peroxidase-enzymes that turn over ROS-increased after HBO treatment, hence the observation of decreased ROS. Since ROS levels are lowered, we explored the effect that this would have on activation of MMP-9. Quantitative analysis with an affinity resin that binds and pulls down the active MMPs exclusively, coupled with proteomics, revealed that HBO treatment indeed reduces the active MMP-9 levels. This work for the first time demonstrates that diminution of active MMP-9 is a contributing factor and a mechanism for enhancement of diabetic wound healing by HBO therapy.

Modification of HIF-1α, NF-aκB, IGFBP-3, VEGF and adiponectin in diabetic foot ulcers treated with hyperbaric oxygen.

Introduction: Diabetic foot ulcers are a frequent complication of diabetes and the first cause of non-traumatic lower limb amputation. They affect quality of life, restrict social productivity and generate a high economic burden for health care systems. Hyperbaric oxygen (HBO2) therapy is an adjunctive treatment option because it improves wound healing in the short term. However, its ability to modulate the pro- and anti-inflammatory balance and the hypoxic cell response in the clinical setting has not been fully described. Objective: To determine modifications in HIF-1α, NF-κB, IGFBP-3, and VEGF expression in wounds as well as circulating inflammatory cytokines in patients with diabetic foot ulcers subjected to HBO2. Materials and methods: We studied 17 ambulatory patients and one hospitalized patient with diabetic foot ulcers classified as Grade 3 or 4 according to the Wagner scale. All underwent HBO2 therapy. Tissue expression of HIF-1α, NF-κB, IGFBP-3, and VEGF was determined by immunohistochemistry. Plasma levels of adiponectin, IL-6, IFN-γ, IL-10 and IL-4 were measured by ELISA and chemiluminescence. Fibrosis and angiogenesis were determined by Masson's trichrome staining. Results: Ulcers in all patients healed after one month of HBO2, and none presented relapses at the one-year follow-up. At the beginning of treatment, HIF-1α and NF-κB expression was observed mainly in the nucleus, whereas these proteins were localized in the cytoplasm at the end of HBO2. There were significant modifications in VEGF expression after therapy, an increase in the plasma level of proinflammatory IL-6, and a decrease in that of IFN-γ. IGFBP-3 expression and plasma levels of adiponectin were increased at the end of HBO2. Increases in fibrosis and angiogenesis were also observed. Conclusion: These results suggest that adjuvant HBO2 modifies the proinflammatory balance related to the cellular response to hypoxia.

Tissue-specific role of Nrf2 in the treatment of diabetic foot ulcers during hyperbaric oxygen therapy.

Hyperbaric oxygen (HBO) therapy is proven to be very successful for diabetic foot ulcer (DFU) treatment due to its antimicrobial effect, increased angiogenesis and enhanced collagen synthesis. The molecular mechanism underlying HBO therapy particularly the involvement of Nrf2 in the wound healing process was investigated in the present study. In addition, we have studied the levels of angiogenic markers in ulcer tissues and their correlation with Nrf2 during HBO therapy compared with standard therapy (Non-HBO) for DFU. A total of 32 Patients were recruited and randomized to standard wound care procedure alone (n = 17) or HBO therapy in combination with standard wound care procedure (n = 15) for 20 days. Our results showed that the tissue levels of Nrf2 along with its downstream targets were significantly increased in patients who underwent HBO therapy when compared to Non-HBO therapy. Further, HBO therapy induced angiogenesis as assessed by increased levels of angiogenesis markers such as EGF, VEGF, PDGF, FGF-2 and CXCL10 in the tissue samples. The expressions of eNOS and nitrite concentrations were also significantly increased in HBO therapy when compared to Non-HBO therapy subjects. Moreover, HBO therapy sensitises the macrophages to release FGF-2 and EGF thereby promotes angiogenesis. Further, it increased the levels of neutrophil attractant CXCL-8 thereby promotes the release of chemokine CCL2, a well-known mediator of neovascularization. The Pearson correlation showed that Nrf2 has a positive correlation with EGF, VEGF and PDGF. In conclusion, the findings of the present study suggest that HBO therapy promotes wound healing by increasing oxygen supply and distribution to damaged tissues, stimulating angiogenesis, decreasing inflammation, and increasing the nitrite levels. Increased levels of Nrf2 transiently regulate the expression of angiogenic genes in wound biopsies, which may result in accelerated healing of chronic wounds.

eNOS Uncoupling: A Therapeutic Target For Ischemic Foot of Diabetic Rat.

AIM: To determine the relationship between eNOS uncoupling and diabetic ischemic foot and whether reversing eNOS uncoupling by Dihydrofolate reductase (DHFR) transfection or folic acid (FA) supplementation can be beneficiary in diabetic ischemic foot. METHODS: The bilateral common iliac artery of diabetic rats were ligated to establish the diabetic ischemic foot animal model. DHFR transfection was implemented via femoral artery and muscle injection of in vivo transfection reagent mixture (GenEscortIII) every 4 days during the 2 weeks intervention. The color doppler flow imaging (CDFI) of femoral artery for RI measurement, triceps and quadriceps structure and histology, eNOS coupling status, DHFR expression level, superoxide, peroxynitrite (ONOO- ) and nitric oxide (NO) production in the presence or absence of L-NAME (eNOS inhibitor) were examined among wild type rats (WT), diabetic sham rats (DM), rats of diabetic ischemic foot (DF) or DF with DHFR transfection (DFT) or DF with FA supplementation (DFF). RESULTS: Dihydroethidium (DHE) fluorescence, as an index of superoxide production was enhanced in the femoral arteries of diabetic rats and even more in those of ischemic foot from diabetic rats. However, the DHE fluorescence was diminished in the presence of L-NAME suggesting eNOS uncoupling is the source of superoxide overproduction which further led to increased peroxynitrite production and decreased NO. bioavailability. Subsequently, the hind limb muscle became atrophic and the local collateral circulation was defective due to endothelial dysfunction related to eNOS uncoupling. However, all of the above and hemodynamic index (RI) of femoral artery were resumed via restoration of DHFR protein expression by folic acid treatment or DHFR transfection. CONCLUSIONS: eNOS uncoupling is involved in diabetic ischemic foot due to DHFR suppression. DHFR restoration can reverse eNOS uncoupling and resume the endothelial dysfunction and pathological changes (increased vasculature resistance, hind limb muscle atrophy and defective collateral circulation) associated with eNOS uncoupling in diabetic ischemic foot. All of which enlightens a novel therapeutic strategy for future diabetic ischemic foot treatments.

Might hyperbaric oxygen therapy (HBOT) reduce renal injury in diabetic people with diabetes mellitus? From preclinical models to human metabolomics.

Diabetic kidney disease (DKD) is the leading cause of end-stage renal failure in the western world. Current treatment of diabetic kidney disease relies on nutritional management and drug therapies to achieve metabolic control. Here, we discuss the potential application of hyperbaric oxygen therapy (HBOT) for the treatment of diabetic kidney disease (DKD), a treatment which requires patients to breathe in 100% oxygen at elevated ambient pressures. HBOT has traditionally been used to diabetic foot ulcers (DFU) refractory to conventional medical treatments. Successful clinic responses seen in the DFU provide the underlying therapeutic rationale for testing HBOT in the setting of DKD. Both the DFU and DKD have microvascular endothelial disease as a common underlying pathologic feature. Supporting evidence for HBOT of DKD comes from previous animal studies and from our preliminary prospective clinical trial reported here. We report urinary metabolomic data obtained from patients undergoing HBOT for DFU, before and after exposure to 6 weeks of HBOT. The preliminary data support the concept that HBOT can reduce biomarkers of renal injury, oxidant stress, and mitochondrial dysfunction in patients receiving HBOT for DFU. Further studies are needed to confirm these initial findings and correlate them with simultaneous measures of renal function. HBOT is a safe and effective treatment for DFU and could also be for individuals with DKD.

Magnesium Supplementation and the Effects on Wound Healing and Metabolic Status in Patients with Diabetic Foot Ulcer: a Randomized, Double-Blind, Placebo-Controlled Trial.

Hypomagnesemia is associated with the development of neuropathy and abnormal platelet activity, both of which are risk factors for diabetic foot ulcer (DFU). This study was carried out to evaluate the effects of magnesium administration on wound healing and metabolic status in subjects with DFU. This randomized, double-blind, placebo-controlled trial was performed among 70 subjects with grade 3 DFU. Subjects were randomly divided into two groups (35 subjects each group) to receive either 250 mg magnesium oxide supplements or placebo daily for 12 weeks. Pre- and post-intervention wound depth and appearance were scored in accordance with the "Wagner-Meggitt's" wound assessment tool. Fasting blood samples were taken at baseline and after the 12-week intervention to assess related markers. After the 12-week treatment, compared with the placebo, magnesium supplementation resulted in a significant increase in serum magnesium (+0.3 ± 0.3 vs. -0.1 ± 0.2 mg/dL, P < 0.001) and significant reductions in ulcer length (-1.8 ± 2.0 vs. -0.9 ± 1.1 cm, P = 0.01), width (-1.6 ± 2.0 vs. -0.8 ± 0.9 cm, P = 0.02), and depth (-0.8 ± 0.8 vs. -0.3 ± 0.5 cm, P = 0.003). In addition, significant reductions in fasting plasma glucose (-45.4 ± 82.6 vs. -10.6 ± 53.7 mg/dL, P = 0.04), serum insulin values (-2.4 ± 5.6 vs. +1.5 ± 9.6 µIU/mL, P = 0.04), and HbA1c (-0.7 ± 1.5 vs. -0.1 ± 0.4%, P = 0.03) and a significant rise in the quantitative insulin sensitivity check index (+0.01 ± 0.01 vs. -0.004 ± 0.02, P = 0.01) were seen following supplementation of magnesium compared with the placebo. Additionally, compared with the placebo, taking magnesium resulted in significant decrease in serum high-sensitivity C-reactive protein (hs-CRP) (-19.6 ± 32.5 vs. -4.8 ± 11.2 mg/L, P = 0.01) and significant increase in plasma total antioxidant capacity (TAC) concentrations (+6.4 ± 65.2 vs. -129.9 ± 208.3 mmol/L, P < 0.001). Overall, magnesium supplementation for 12 weeks among subjects with DFU had beneficial effects on parameters of ulcer size, glucose metabolism, serum hs-CRP, and plasma TAC levels. CLINICAL TRIAL REGISTRATION NUMBER: http://www.irct.ir : IRCT201612225623N96.

Curcumin induces therapeutic angiogenesis in a diabetic mouse hindlimb ischemia model via modulating the function of endothelial progenitor cells.

BACKGROUND: Neovascularization is impaired in diabetes mellitus, which leads to the development of peripheral arterial disease and is mainly attributed to the dysfunction of endothelial progenitor cells (EPCs). Previous studies proved the promotional effect of curcumin on neovascularization in wound healing of diabetes. Thus, we hypothesize that curcumin could promote neovascularization at sites of hindlimb ischemia in diabetes and might take effect via modulating the function of EPCs. METHODS: Streptozotocin-induced type 1 diabetic mice and nondiabetic mice both received unilateral hindlimb ischemic surgery. Curcumin was then administrated to the mice by lavage for 14 days consecutively. Laser Doppler perfusion imaging was conducted to demonstrate the blood flow reperfusion. Capillary density was measured in the ischemic gastrocnemius muscle. In addition, angiogenesis, migration, proliferation abilities, and senescence were determined in EPCs isolated from diabetic and nondiabetic mice. Quantitative PCR was then used to determine the mRNA expression of vascular endothelial growth factor (VEGF) and angiopoetin-1 (Ang-1) in EPCs. RESULTS: Curcumin application to type 1 diabetic mice significantly improved blood reperfusion and increased the capillary density in ischemic hindlimbs. The in-vitro study also revealed that the angiogenesis, migration, and proliferation abilities of EPCs and the number of senescent EPCs were reversed by curcumin application. Quantitative PCR confirmed the overexpression of VEGF-A and Ang-1 in EPCs after curcumin treatment. CONCLUSION: Curcumin could enhance neovascularization via promoting the function of EPCs in a diabetic mouse hindlimb ischemia model.

Amputation predictors in diabetic foot ulcers treated with hyperbaric oxygen.

OBJECTIVE: Although hyperbaric oxygen therapy (HBOT) has long been used for diabetic foot ulcers (DFUs), its effectiveness is still controversial. The aim of this study was to investigate the efficacy of HBOT in the management of DFUs and identify amputation predictors. METHOD: Patients with chronic DFUs (Wanger grade 2-5) were included in the study, which took place between January 2010 and December 2012. HBOT, 100% oxygen, 2.4 atmosphere absolute (ATA) for 120 minutes, was administered to all patients in addition to standard treatment. DFUs were monitored for at least 3 years, or until healing or amputation occurred. RESULTS: Patients with a total of 146 chronic DFUswere recruited. Complete healing (69.6%) and significant improvement (17.9%) was observed in 87.5% of the patients. The cases with no improvement resulted in amputation (minor amputation: 15.0%; major amputation: 8.2%). The duration of diabetes (p=0.037), new wound formation (p=0.045), C-reactive protein (p=0.001) and Wagner grade (p=0.0001) were correlated with amputation in multiple regression analysis. Mortality was higher in the amputation group than in the non-amputation group (47.1 % versus 21.4 %, p=0.007). CONCLUSION: The inclusion of HBOT with standard treatment and a multidisciplinary approach may be useful in the treatment of DFUs. We found the most important predictors of amputation to be Wagner grade and wound infection. Multicentre, prospective, randomised studies are needed to provide more evidence.

Clinical and metabolic response to flaxseed oil omega-3 fatty acids supplementation in patients with diabetic foot ulcer: A randomized, double-blind, placebo-controlled trial.

BACKGROUND: Data on the effects of flaxseed oil omega-3 fatty acids supplementation on wound healing and metabolic status in subjects with diabetic foot ulcer (DFU) are scarce. OBJECTIVE: This study was conducted to evaluate the effects of flaxseed oil omega-3 fatty acids supplementation on wound healing and metabolic status in subjects with DFU. METHODS: The current randomized, double-blind, placebo-controlled trial was conducted among 60 subjects (aged 40-85years old) with grade 3 DFU. Subjects were randomly allocated into two groups (30 subjects each group) to receive either 1000mg omega-3 fatty acids from flaxseed oil supplements or placebo twice a day for 12weeks. RESULTS: After the 12-week intervention, compared with the placebo, omega-3 fatty acids supplementation resulted in significant decreases in ulcer length (-2.0±2.3 vs. -1.0±1.1cm, P=0.03), width (-1.8±1.7 vs. -1.0±1.0cm, P=0.02) and depth (-0.8±0.6 vs. -0.5±0.5cm, P=0.01). Additionally, significant reductions in serum insulin concentrations (-4.4±5.5 vs. +1.4±8.3 µIU/mL, P=0.002), homeostasis model of assessment-estimated insulin resistance (-2.1±3.0 vs. +1.0±5.0, P=0.005) and HbA1c (-0.9±1.5 vs. -0.1±0.4%, P=0.01), and a significant rise in the quantitative insulin sensitivity check index (+0.01±0.01 vs. -0.005±0.02, P=0.002) were seen following supplementation with omega-3 fatty acids compared with the placebo. In addition, omega-3 fatty acids supplementation significantly decreased serum high sensitivity C-reactive protein (hs-CRP) (-25.5±31.5 vs. -8.2±18.9µg/mL, P=0.01), and significantly increased plasma total antioxidant capacity (TAC) (+83.5±111.7 vs. -73.4±195.5mmol/L, P<0.001) and glutathione (GSH) concentrations (+60.7±140.2 vs. -15.5±129.7µmol/L, P=0.03) compared with the placebo. CONCLUSIONS: Overall, omega-3 fatty acids supplementation for 12weeks among subjects with DFU had beneficial effects on parameters of ulcer size, markers of insulin metabolism, serum hs-CRP, plasma TAC and GSH levels. In addition, flaxseed oil omega-3 fatty acids may have played an indirect role in wound healing due to its effects on improved metabolic profiles.

Preparation and in vivo evaluation of cationic elastic liposomes comprising highly skin-permeable growth factors combined with hyaluronic acid for enhanced diabetic wound-healing therapy.

To enhance the therapeutic effects of exogenous administration of growth factors (GFs) in the treatment of chronic wounds, we constructed GF combinations of highly skin-permeable epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), and platelet-derived growth factor-A (PDGF-A). We genetically conjugated a low-molecular-weight protamine (LMWP) to the N-termini of these GFs to form LMWP-EGF, LMWP-IGF-I, and LMWP-PDGF-A. Subsequently, these molecules were complexed with hyaluronic acid (HA). Combinations of native or LMWP-fused GFs significantly promoted fibroblast proliferation and the synthesis of procollagen, with a magnification of these results observed after the GFs were complexed with HA. The optimal proportions of LMWP-EGF, LMWP-IGF-I, LMWP-PDGF-A, and HA were 1, 1, 0.02, and 200, respectively. After confirming the presence of a synergistic effect, we incorporated the LMWP-fused GFs-HA complex into cationic elastic liposomes (ELs) of 107±0.757nm in diameter and a zeta potential of 56.5±1.13mV. The LMWP-fused GFs had significantly improved skin permeation compared with native GFs. The in vitro wound recovery rate of the LMWP-fused GFs-HA complex was 23% higher than that of cationic ELs composed of LMWP-fused GFs alone. Moreover, the cationic ELs containing the LMWP-fused GFs-HA complex significantly accelerated the wound closure rate in a diabetic mouse model and the wound size was maximally decreased by 65% and 58% compared to cationic ELs loaded with vehicle or native GFs-HA complex, respectively. Thus, topical treatment with cationic ELs loaded with the LMWP-fused GFs-HA complex synergistically enhanced the healing of chronic wounds, exerting both rapid and prolonged effects. STATEMENT OF SIGNIFICANCE: We believe that our study makes a significant contribution to the literature, because it demonstrated the potential application of cationic elastic liposomes as topical delivery systems for growth factors (GFs) that have certain limitations in their therapeutic effects (e.g., low percutaneous absorption of GFs at the lesion site and the requirement for various GFs at different healing stages). Topical treatment with cationic elastic liposomes loaded with highly skin-permeable low-molecular-weight protamine (LMWP)-fused GFs-hyaluronic acid (HA) complex synergistically enhanced the healing of diabetic wounds, exerting both rapid and prolonged effects.

The effects of zinc supplementation on wound healing and metabolic status in patients with diabetic foot ulcer: A randomized, double-blind, placebo-controlled trial.

This study was performed to determine the effects of zinc supplementation on wound healing and metabolic status in patients with diabetic foot ulcer. The current randomized, double-blind, placebo-controlled trial was conducted among 60 patients (aged 40-85 years old) with grade 3 diabetic foot ulcer. Participants were randomly divided into two groups (30 participants in each group) to take either 220 mg zinc sulfate supplements containing 50 mg elemental zinc or placebo daily for 12 weeks. After the 12-week intervention, compared with the placebo, zinc supplementation was associated with significant reductions in ulcer length (-1.5 ± 0.7 vs. -0.9 ± 1.2 cm, p = 0.02) and width (-1.4 ± 0.8 vs. -0.8 ± 1.0 cm, p = 0.02). In addition, changes in fasting plasma glucose (-40.5 ± 71.0 vs. -3.9 ± 48.5 mg/dl, p = 0.02), serum insulin concentration (-8.0 ± 15.4 vs. +1.1 ± 10.3 µIU/ml, p = 0.009), homeostasis model of assessment-estimated insulin resistance (-3.9 ± 7.1 vs. +0.8 ± 5.9, p = 0.007), the quantitative insulin sensitivity check index (+0.01 ± 0.03 vs. -0.002 ± 0.02, p = 0.04) and HbA1c (-0.5 ± 0.8 vs. -0.1 ± 0.5%, p = 0.01) in the supplemented group were significantly different from the changes in these indicators in the placebo group. Additionally, significant increases in serum HDL-cholesterol (+4.1 ± 4.3 vs. +1.1 ± 5.1 mg/dl, p = 0.01), plasma total antioxidant capacity (+91.7 ± 213.9 vs. -111.9 ± 188.7 mmol/L, p < 0.01) and total glutathione (+68.1 ± 140.8 vs. -35.0 ± 136.1 µmol/L, p = 0.006), and significant decreases in high sensitivity C-reactive protein (-20.4 ± 24.6 vs. -6.8 ± 21.3 µg/ml, p = 0.02) and plasma malondialdehyde concentrations (-0.6 ± 0.9 vs. -0.2 ± 0.7 µmol/L, p = 0.03) were seen following supplementation with zinc compared with the placebo. Zinc supplementation for 12 weeks among diabetic foot ulcer patients had beneficial effects on parameters of ulcer size and metabolic profiles.

The effects of vitamin D supplementation on wound healing and metabolic status in patients with diabetic foot ulcer: A randomized, double-blind, placebo-controlled trial.

OBJECTIVE: This study was conducted to evaluate the effects of vitamin D supplementation on wound healing and metabolic status in patients with diabetic foot ulcer (DFU). METHODS: This randomized, double-blind, placebo-controlled trial was performed among 60 patients with grade 3 DFU according to "Wagner-Meggitt's" criteria. Participants were randomly divided into two groups (each 30 participants) and received either 50,000IU vitamin D supplements every 2weeks for 12weeks (group A) or placebo (group B). Fasting blood samples were taken at study baseline and after 12-week intervention to determine related markers. RESULTS: After 12weeks of intervention, compared with the placebo, vitamin D supplementation resulted in a significant reduction in ulcer length (-2.1±1.1 vs. -1.1±1.1cm, P=0.001), width (-2.0±1.2 vs. -1.1±1.0cm, P=0.02) and depth (-1.0±0.5 vs. -0.5±0.5cm, P<0.001), and erythema rate (100% vs. 80%, P=0.01). In addition, in supplemented patients changes in serum insulin concentration (-3.4±9.2 vs. +2.8±9.3 µIU/mL, P=0.01), homeostasis model of assessment-estimated insulin resistance (-1.5±4.1 vs. +1.7±5.1, P=0.01), the quantitative insulin sensitivity check index (+0.006±0.02 vs. -0.006±0.02, P=0.03) and HbA1c (-0.6±0.6 vs. -0.1±0.5%, P=0.004) were significantly different from those of patients in the placebo group. Additionally, following supplementation with vitamin D, significant reductions in serum total- (-15.8±18.9 vs. +5.3±31.8mg/dL, P=0.003), LDL- (-17.2±19.8 vs. +2.2±28.6mg/dL, P=0.003), total-/HDL-cholesterol ratio (-1.1±0.8 vs. -0.2±1.1, P=0.001), high sensitivity C-reactive protein (hs-CRP) (-0.4±2.5 vs. +1.9±4.2µg/mL, P=0.01), erythrocyte sedimentation rate (ESR) (-34.7±32.4 vs. -18.0±26.6mm/h, P=0.03) and plasma malondialdehyde (MDA) concentrations (-0.7±0.9 vs. -0.2±0.5µmol/L, P=0.008) were seen compared with the placebo. CONCLUSIONS: Overall, vitamin D supplementation for 12weeks among patients with DFU had beneficial effects on glucose homeostasis, total-, LDL-, total-/HDL-cholesterol, ESR, hs-CRP and MDA levels. In addition, vitamin D may have played an indirect role in wound healing due to its effect on improved glycemic control.

Increasing the miR-126 expression in the peripheral blood of patients with diabetic foot ulcers treated with maggot debridement therapy.

BACKGROUND: miR-126 may increase angiogenesis in patients with diabetic foot ulcers (DFUs) treated with maggot debridement therapy (MDT). METHODS: Real-time quantitative PCR was used to detect expression of miR-126 mRNA in the peripheral blood among the non-diabetic population, type 2 diabetes mellitus patients without DFU, and patients with DFUs of type 2 diabetes mellitus. The expression of miR-126 mRNA in the peripheral blood of patients with DFUs was observed before and after MDT. Finally, human umbilical vein endothelial cells (HUVEC) were utilized to explore miR-126 mRNA expression with maggot excretions/secretions (ES). RESULTS: In the patients with DFUs, the miR-126 mRNA expression level in the peripheral blood was less than that type 2 diabetes mellitus patients without DFU, and much lower than that in the non-diabetic population (P<0.001). The miR-126 expression level was significantly increased in those DFU patients treated with MDT (P<0.05). Finally, using HUVEC co-cultured with ES, we showed the ES increased miR-126 expression in vitro (P<0.001). CONCLUSION: MDT upregulates the miR-126 expression in the peripheral blood of patients with DFUs.